Abstract

Aim: To
evaluate the antifertility activity of various extracts of Crotalaria
juncea seeds in male mice. Methods: Adult male mice were gavaged
the petroleum ether, benzene and ethanol extracts of C.juncea
seeds, 25 mg·(100g)-1·day-1
for 30 days. On day 31 the animals were sacrificed by cervical dislocation
and the testes, epididymis, vas deferens, seminal vesicles, prostate gland,
bulbourethral gland and levator ani were dissected out and weighed. The
organs were processed for biochemical and histological examination. Results:
In petroleum ether, benzene and ethanol extracts treated rats, there was
a decrease in the weights of testis and accessory reproductive organs.
The diameters of the testis and seminiferous tubules were decreased. Spermatogonia,
spermatocytes and spermatids in the testis and the sperm count in cauda
epididymis were also decreased. There was a significant reduction in the
protein and glycogen contents and an increase in the cholesterol content
in the testis, epididymis and vas deferens. Of the 3 extracts, the ethanol
extract appeared to be the most potent in antispermatogenic activity.
When the ethanol extract was tested in immature male mice, there was an
antiandrogenic effect as the weights of accessory organs were reduced.
Conclusion: The various extracts of C. juncea seeds arrest
spermatogenesis and are likely to have an antiandrogenic activity.

1 Introduction

Plants have served as a natural source of antifertility substances. Henshaw
[1] has listed many plants used by primitive people in different countries
to control fertility. Though many indigenous plants have been shown to
prevent birth, only a few have so far been investigated for antispermatogenic
activity [2-5]. Crotalaria juncea Linn. (Papilionaceae), commonly
known as Sunn hemp, is cultivated throughout India and Indian Ayurvedic
medicine indicated that various parts of C. juncea have analgesic,
astringent, emmenagogue and abortifacient activities and is also used
for the treatment of skin diseases. The seeds of this plant have been
reported to possess contraceptive activity [6], however, no detailed study
is available so far on its possible male antifertility activity. The present
study was designed to clarify this point in albino mice.

2 Materials and
methods

2.1 Plant material

The fresh seeds of C. juncea were obtained from
a local source during October and November 2000 and were authenticated
by Dr. Y. N. Sitharam, Department of Botany, Gulbarga University, Gulbarga
(HGUG No. 71), India, where voucher specimens were deposited.

2.2 Extract preparation

The seeds were shade-dried,
powdered and subjected to Soxhlet extraction successively and separately
with petroleum ether (B. P. 60-80 ),
benzene and ethanol (95 %). The decoctions produced were evaporated under
reduced pressure below 45 .
The residual extracts thus obtained (500 g of C. juncea seed produces:
3.8 g of petroleum ether extract, 1.2 g of benzene extract and 2.73 g
of ethanol extract) were screened for their antifertility activity in
mice. Before use, the extract was suspended in Tween-80 (1 %) normal saline
with a final concentration of 25 mg/mL.

2.3 Animals

Adult, healthy and virgin Swiss strain male albino mice
(Mus musculus), 60-70 days old and 35-40 g body weight, were selected
from the inbred animal colony for experimental use. The animals were maintained
under uniform husbandary conditions of light and temperature and were
given pellet diet (Central Food and Technological Research Institute,
Mysore, India) and tap water ad libitum. The animals were divided
into 4 groups of 6 each.

2.4 Treatment

After preliminary trials, a dose of 25 mg/100 g body
weight per day ip for 30 days was determined for evaluating the antifertility
effect. Group 1 served as the control and received the vehicle. Group
2, 3 and 4 received the petroleum ether, the benzene and the ethanolic
extract in Tween-80, respectively.

2.5 Observations

The animals were sacrificed 24 h after the last treatment.
The testes, epididymis, seminal vesicles, vas deferens, prostate, bulbourethral
gland, and levator ani were excised, blotted free of blood, carefully
freed from the surrounding fat and connective tissue and weighed up to
the nearest mg on an electronic balance. Fresh tissues from testis, epididymis
and vas deferens were processed for the estimation of glycogen, protein
and cholesterol by conventional methods. Besides, they were fixed in Bouin's
fluid, embedded in paraffin, sectioned at 5 mm and stained with haematoxylin-eosin
for histological examination. The testicular and seminiferous tubular
diameters were calculated by the method described by Deb et al
[7]. Spermatogenic element count was made from randomly chosen twenty
round cross-sections. The cauda epididymal sperm suspension was prepared
in normal saline and epididymal sperm count was estimated by the method
of Kempinas and Lamano Carvalho [8].

2.6 Antiandrogenic activity

Swiss strain immature mice of 25 days old were used to
assess the possible antiandrogenic activity. Among the 3 extracts, the
ethanolic extract showed the maximum changes in 2.5 (Observation). Thus
it was used in this experiment with 3 groups of 6 animals each: Group
A: Vehicle control, Group B: 20 mg testosterone ip per animal per day
for 5 days, and Group C: Ethanol extract25 mg/100 g body weight
per day ip for 5 days.

All the animals were sacrificed on day 6 by cervical
dislocation and the testes, epididymis, seminal vesicles, vas deferens,
prostate, bulbourethral gland and levator ani were excised, blotted free
of blood, carefully freed from surrounding fat and connective tissue and
weighed up to the nearest mg.

2.7 Statistical analysis

The data were expressed as meanSE and Student's
t test was used to assess the statistical significance.

3 Results

3.1 Changes in testis

3.1.1 Gravimetric and histometric
changes

A significant (P<0.01) reduction
in the weight of testis was observed in the ethanol, petroleum ether and
benzene extracts groups. Histometric data were similar to those of weight
changes: a significant (P<0.01) reduction in the diameters of
the testis and seminiferous tubules with all the 3 extracts (Table 1).

The number of spermatogonia, spermatocytes
and spermatids are significantly reduced (P<0.01) with the ethanol
extract, while the petroleum ether and benzene extracts significantly
reduced the number of spermatogonia and spermatocytes (P<0.01).
In all treated groups, there were pyknosis in the primary and secondary
spermatocytes and degeneration of Leydig cells, spermatozoa were completely
absent from the seminiferous tubular lumen and the sperm density was significantly
reduced in the cauda epididymis (P<0.01) (Table 1).

3.1.3 Biochemical changes

The cholesterol content of the testis
was significantly (P<0.01) increased in the ethanol and benzene
extract groups, while the protein content was significantly reduced in
the ethanol extract group (P<0.01) and in the petroleum ether
and benzene extract groups (P<0.05). The glycogen content was
significantly reduced with the benzene and ethanol extracts (P<0.01),
but not with the petroleum ether extract (Table 1).

3.2 Changes in accessory organs

3.2.1 Epididymis

The weight of epididymis was significantly
reduced in the ethanol and benzene groups (P<0.01), but not
in the petroleum ether group. The cholesterol content of the epididymis
was significantly increased (P<0.01) and the protein and glycogen
contents of epididymis were significantly reduced (P<0.01) with
all the three extracts (Tables 2 & 3).

Table 3. Effect of various extracts of C.
juncea seeds on epididymal and vasal biochemistry (meanSE).
cP<0.01, compared with controls.

Group
(n=6)

Epididymis

Vas
deferens

Cholesterol(µg/mg)

Protein
(µg/mg)

Glycogen
(µg/mg)

Protein
(µg/mg)

Glycogen
(µg/mg)

1
(Control)

1.760.24

1.280.15

1.540.14

1.010.09

2.370.26

2
(Petroleum ether
extract)

2.360.20c

0.470.01c

0.760.02c

0.760.04

1.720.02

3
(benzene
extract)

2.080.15c

0.480.09c

0.740.03c

0.700.03

1.680.03

4
(ethanol
extract)

2.430.01c

0.460.02c

0.730.01c

0.680.03c

1.66
0.22c

3.2.2 Vas deferens

A significant reduction in the weight
of vas deferens (P<0.01) was observed in the ethanol extract
group, but not in the other 2 groups. The protein content of vas deferens
was significantly reduced (P<0.01) with the ethanol extract,
whereas insignificant with petroleum ether and benzene extracts. Though
all the 3 extracts reduced the glycogen content of vas deferens, it was
significant (P<0.01) only with the ethanol extract (Tables 2
& 3).

3.2.3 Seminal vesicles and prostate

The weight of seminal vesicle was
significantly (P<0.01) reduced with all the three extracts.
The weight of prostate was significantly reduced (P<0.01) with
the ethanol extract, but not with the petroleum ether and benzene extracts
(Table 2).

3.2.4 Bulbourethral gland and levator
ani

The weights of the bulbourethral gland
and levator ani showed a significant (P<0.01) reduction in the
benzene, ethanolic and petroleum ether groups (Table 2).

3.3 Antiandrogenic activity

Testosterone administration caused a highly significant
(P<0.01) increase in the wet weights of epididymis, vas deferens,
seminal vesicles, prostate, bulbourethral gland and levator ani. On the
contrary, the ethanol extract significantly decreased the wet weights
of these organs (P<0.01) compared to the controls, indicating
an antiandrogenicity of the ethanol extract(Table 4).

Group
(n=6)

A(Control)

B(Testosterone)

635.61.3c

208.20.9c

31.60.5c

312.00.7c

96.20.5c

101.40.2c

99.20.1c

C(Ethanol
extract)

619.50.2

113.90.2c

18.20.4c

170.40.2c

41.50.2c

44.50.2c

51.00.3c

4 Discussion

Administration of the petroleum ether,
benzene and ethanol extracts of C. juncea reduces the testicular
weight, which may be due to a decreased production of seminiferous tubular
fluid that contributes to the weight of testis [9]. The reduced protein
content may be another reason as the growth rate of any organ is proportional
to its protein content. The observed reduction in the number of spermatogonia,
spermatocytes and spermatids may indicate lowered availability of FSH
and LH/ICSH, which are essential for initiation and maintenance of spermatogenesis.
For this reason, the seminiferous tubular lumen is devoid of spermatozoa.

The glycogen content in the cell represents
the energy storage. The Sertoli cells and spermatogonia contain glycogen
and provide nourishments to the seminiferous tubular cells and the glycogen
content is found to be directly proportional to the steroid hormone levels
[10]. A decrease in the glycogen content of the testis reduces the energy
source for spermatogenic activity.

The increased cholesterol content of testis may reflect
a reduced conversion of cholesterol to androgens, which is dependent on
the availability of LH/ ICSH. The reduction or non-availability of androgens
is further supported by the reduction in the weight of accessory organs.
All these organs play important roles in sperm maturation and mobility
and the formation of semen. The ethanol extract when tested in immature
mice also showed antiandrogenic effect. In conclusion, various extracts
of C. juncea seeds arrest spermatogenesis and are likely to have
an antiandrogenic activity.